When performing genetic analysis, there is generally a need to amplify the number of copies in the sample, as the number present in the sample is generally too few to be detected.
This can be done using, for example, thermocycling or isothermal amplification.
Isothermal techniques include SDA, LAMP, SMAP, ICAN, SMART. The reaction proceeds at a constant temperature using strand displacement reactions. Amplification can be completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with strand displacement activity, and substrates at a constant temperature. In one technique, called Loop-mediated isothermal amplification (LAMP), target-specific amplification is achieved by the use of 4 to 6 different primers specifically designed to recognize 6 to 8 distinct regions on the target gene, respectively. LAMP is further described in Eiken Chemical's patent EP2045337 ‘Process for synthesizing nucleic acid’, incorporated here by reference.
Such methods typically amplify nucleic acid copies 109-1010 times in 15-60 minutes.
In addition to the primers, strand displacement techniques use Tris and sulphate compounds (such as MgSO4, NH4SO4) to maintain enzyme functionality.
Tris is an organic compound (more formally known as tris (hydroxymethyl) aminomethane, with the formula (HOCH2)3CNH2). Strand displacement techniques, such as LAMP, use Tris as a buffer, which maintain the reaction at the optimal pH.
The recommended concentration of Tris and Sulphates is 20 mM or more and 12-20 mM respectively.
Once the nucleic acid is amplified, a nucleic acid assay requires a secondary detection technology such as spectrophotometry, turbidity, LFD (lateral flow dipsticks) or luciferase. However, such known techniques have drawbacks. Fluorescent reagents require labelling to allow UV fluorescence, making it expensive. Furthermore, reagents such as SYBR green binds to DNA making it inherently carcinogenic; the Ames Test shows it to be both mutagenic and cytotoxic. Also SYBR green is not specific and attaches to any double stranded DNA thus increasing background signal. Turbidity measurements require expensive instrumentation to provide quantification. Lastly, the reagents used in LFD require secondary conjugation which is susceptible to non-specific detection.
The existing isothermal techniques are not suitable for systems employing pH detection. Thus there is a need in the art for a kit and method for isothermally amplifying nucleic acids and efficiently detecting them with a safe inexpensive device. Surprisingly, the inventors have found that the reagents used may increase the yield of amplification.